Law enforcement officials, workplace drug testing programs, and researchers in the field of drug abuse lack rapid, easily deployable tools to measure present cannabis (marijuana) intoxication. The enzyme-linked immunosorbant assay (ELISA) is an established cannabis testing method for detecting delta-9-tetrahydrocannabinol (THC) levels in urine or blood; however, the logistics of obtaining urine or blood samples on site represents a major limitation of current THC ELISAs, particularly for curbside testing of drivers. The most feasible candidate sample for curbside testing is the oral fluid, as it is a minimally invasive sampling route and the pharmacokinetics of THC concentration in oral fluid (half-life of 1.5 hours) parallels that of serum; however, the primary obstacle to this approach is the lack of a rapid, quantitative THC immunoassay. Traditional plate-based ELISA tests can take 1 to 3 hours and require a laboratory setting, and lateral flow assays lack the accuracy needed for quantitative cannabis intoxication detection. BreviTest Technologies LLC is developing a portable analyzer to perform a rapid, quantitative ELISA test within a 10-minute run time. The sample is loaded into a discontinuous microfluidics cartridge, and a robotic mechanism controls the movement of antibody-conjugated magnetic nanoparticles through successive fluid wells, where enhanced mixing patterns substantially reduce the time required for each ELISA step. This innovative approach enables the implementation of a rapid, portable, fully automated ELISA test with accuracy comparable to a standard laboratory ELISA. In this SBIR Phase I project, BreviTest will partner with Cayman Chemical Company to develop a THC ELISA test using the BreviTest robotic analyzer, with the following Specific Aims: 1) Develop and produce discontinuous microfluidic cartridges with layman-operable sample port. The type and quantity of magnetic nanoparticles will be determined for optimal reagent mixing and transit parameters, and the design and validation will be completed for a sampling tool for metering 20 L of sample fluid. Design of the cartridge will be optimized for stability, consistency, and compatibility with biological materials. 2) Develop curbside ELISA test to quantitatively measure THC in oral fluid. BreviTest will collaborate with Cayman Chemical to screen commercially available clones to generate an optimized antibody for THC detection. Antibodies will be conjugated to magnetic nanoparticles via streptavidin-biotin linkages, for use in the BreviTest analyzer. Optimization of reagent concentrations and analyzer parameters will be performed, along with statistical validation of dynamic range, specificity, and sensitivity. The completion of these proof-of-concept studies will demonstrate the ability of the BreviTest portable ELISA analyzer to measure THC concentration in oral fluid samples, at concentrations relevant to curbside intoxication analysis. This work will lay the foundation for further Phase II studies including fied and clinical testing of a rapid THC intoxication assay and additional validation testing towards a defined 510(k) clearance by the FDA.